High intestinal parasite infection detected in children from Región Autónoma Atlántico Norte (R.A.A.N.) of Nicaragua

There is a lack of epidemiological information concerning intestinal parasitic infections, and especially in soil-transmitted helminths, occurring in some departments of Nicaragua. Up to now, this is the first study involving two nearby areas (Puerto Cabezas and Siuna municipalities) of the Región Autónoma Atlántico Norte (R.A.A.N.). One stool sample was analyzed by Kato-Katz, formaldehyde-ethyl acetate concentration method and modified Ziehl–Neelsen technique, and a simple questionnaire concerning demographic, sanitary and behavioral data was distributed among 735 children and evaluated. Overall prevalence of infection reached 97.0%, being the highest prevalences detected in all Nicaragua. The higher protozoan prevalence appears in Siuna (94.5%), a rural interior municipality, with a typical tropical monsoon climate, while the higher helminths rates were reached in Puerto Cabezas (92.8%), the urbanized coastal capital, with a typical tropical rainforest climate. No statistical differences were found with regard to sex. However, the 6–11-year age-group children presented the highest prevalences. Most T. trichiura infections (59.4%) were of light intensity, while 51.7% of Ascaris lumbricoides were of moderate intensity. Multivariable logistic regression analysis indicated that those who drink rainwater and walk barefoot were 2.9 and 2.5 times more likely to have helminth infections, respectively. Results from one geographical setting might not be applied to other nearby with different climatic conditions. The use of anthelmintic drugs only will not be sufficient to bring prevalence to low levels. It is necessary to design geographically more specific intervention, with communication and interaction between different disciplines (e.g. parasitology, biochemistry, molecular biology, epidemiology, public health, etc.) being imperative to reduce STH infection.

www.nature.com/scientificreports/ Nicaragua is the largest country of Central America, with approximately 6 million inhabitants, occupying a landmass of 130,967 km2, and being the second poorest country in the Latin American and Caribbean (LAC) region. This country is made up of three regions: the Pacific region, with seven departments; the Center region, with eight departments; and the Atlantic/Caribbean region including the Región Autónoma Atlántico Norte (R.A.A.N.) and the Región Autónoma Atlántico Sur (R.A.A.S.). In the 1990s, Nicaragua established massive deworming programs, with a joint effort of various actors (ministries, NGOs, companies, etc.) in such a way that an 87% coverage of the child population was reported in 2011, and nowadays deworming is included as a new category on child vaccination cards 13 .
Several studies have been conducted on the prevalence of intestinal parasitic infections in Nicaragua [14][15][16][17][18][19][20][21] , although this is the first study published in SCI journals involving R.A.A.N. The goal of this epidemiological study was to determine the prevalence of intestinal parasite infections and the intensity of STH, assessing which variables (demographic/sanitary/behavioral) among schoolchildren were related to the infection, providing the baseline for appropriate strategies against STH infections.

Methods
Study area and climatic conditions. R.A.A.N. covers the largest area (33,105 km 2 ) of Nicaragua, covering 26.3% of the national territory (Fig. 1A). It borders Honduras to the north and has a long coastline along the Caribbean Sea. R.A.A.N., with an estimated population of around 490,390 inhabitants, is divided into eight municipalities (Fig. 1B). The administrative capital is the municipality of Puerto Cabezas (lat. 14°03′N, long. 83°22′W) with around 113,534 inhabitants and 63% from urban areas. The second largest municipality is Siuna (lat. 13°44′N, long. 84°46′W), with around 103,139 inhabitants and 84% belonging to rural areas. In general, homes in rural areas of both municipalities tend to be more dispersed, being surrounded by grassland, without any urbanization and without sanitation.
According to the Köppen-Geiger climate classification 22 , the Pacific region in Nicaragua shows a tropical savanna climate (with a pronounced dry season while the Atlantic region have: a) a tropical monsoon climate (Am), with 2400 to 3000 mm annual rainfall, temperature oscillating between 24 °C and 29 °C, with a winter or rainy season (from May to January) and a summer or dry season (from February to April) with only sporadic rains, typical conditions of the municipality of Siuna; and b) a tropical rainforest climate (Af), with 2000 to 6000 mm annual rainfall, temperature oscillating between 27 °C and 39 °C, 80%-90% relative humidity, with a rainy season only (Fig. 1C), typical conditions of the municipality of Puerto Cabezas.
Population and sample collection. The

Results
Total infections. Table 1 summarizes the total infection results obtained in R.A.A.N., stratified by municipalities. The entire spectrum was made up to 10 protozoa and 6 helminth species, although not all parasite species were found in both municipalities. The isolated cases of Balantidium coli detected in Puerto Cabezas municipality and of Cryptosporidium sp., Taenia sp. and Strongyloides stercoralis detected in Siuna municipality are noteworthy.
Overall prevalence of infection, with at least one parasite species, reached 97.0% in the children surveyed, either being protozoa (90.7%) or helminths (61.6%). Among the R.A.A.N. children surveyed, protozoan infections were more prevalent than helminths, with statistical differences (p < 0.0001).

Gender, age groups and urban/rural area infections.
Prevalence results according to gender, age groups and urban/rural school area are shown in Table 2. No statistical differences were found with regard to gender neither in the total prevalence rates nor within each municipality.
The distribution of total infection prevalence according to age groups revealed significant differences. Total prevalence increased, always being highest in the group of 6-11 years-old children, both in Puerto Cabezas (p = 0.006) and Siuna (p = 0.036) municipalities. However, protozoa infection in Siuna municipality was higher in the adolescent group with statistical differences (p = 0.019) and it was higher in the municipality of Puerto Cabezas in the youngest age group with statistical differences (p = 0.049).
The total infection rate of helminths in urban schools was significantly higher than in rural schools (p = 0.0003). In particular, A. lumbricoides prevalence rates obtained in urban schools were higher than those obtained in rural schools (p < 0.0001), while Hookworm prevalence rates were higher in rural schools (p = 0.005).    The extent of polyparasitism in the entire study according to gender, age groups and urban/rural school area is shown in Fig. 2. There were no statistical differences between females and males or between urban and rural schools. However, polyparasitism was lower (57.3%), with statistical differences (p < 0.0001), among the youngest children studied.  (Table 3). Table 4 shows the prevalence of each class of infection intensity obtained in the two municipalities, according to gender, age group and urban/rural schools studied. Among those infected, most T. trichiura infections were light intensity (59.4%), while 51.7% of A. lumbricoides were moderate-intensity infections. No differences in infection intensity classes appeared among the two municipalities studied in any STH species.
Hookworm infection intensity classes were of light infection intensity (100%) independent of municipality, sex, age group and urban/rural schools studied. In the case of T. trichiura, light-infection intensity in girls and  www.nature.com/scientificreports/ in the urban schools was higher with statistical differences than in boys (p = 0.003) and in the rural schools (p = 0.0007). In the case of A. lumbricoides, moderate and heavy infection intensities were higher, with statistical differences (p < 0.0001 and p = 0.003, respectively) in the 6-11-year-old age group. Table 5 shows the frequency of STH infection grouping. Infection with two STH species is the most frequent association found in R.A.A.N., with the association of T. trichiura and A. lumbricoides standing out.
Only single Hookworm infections appear more frequently in Siuna municipality, with significant differences (p = 0.001). However, double and triple STH infections appear more frequently in Puerto Cabezas than in Siuna municipality, with significant differences (p < 0.0001 and p = 0.023, respectively). Significant positive associations were reached in the co-infection analysis of T. trichiura with respect to other STH detected in this study, but no associations were observed between A. lumbricoides and Hookworm co-infections.

Univariable and multivariable analyses of factors related to infections.
We assessed the association between infection and several variables to determine their impact on the intestinal parasite infection. During univariable analysis, 3 variables were identified with p value < 0.05 in relation to any infection status, such as children's age, source of drinking water and habit of wearing shoes ( Table 6). The odds of helminth infection were highest among 6-11-year-old children, in those drinking rain water (p < 0.0001) and in those walking barefoot (p = 0.025). On the other hand, the odds of helminth infection were lowest among children of the urban schools (p < 0.0001).
The variables with p < 0.05 in the univariable analysis were analyzed using multivariable logistic regression model using the backward stepwise method to assess the helminth infection ( Table 7). The variable urban/rural school area was left out with p value = 0.06. The model identified that the risk of helminth infection is multiplied by 2.6 if the child is 6-11 years old, by 2.9 if the child does not drink safe water, and by 2.5 if the child walks barefoot.

Discussion
The very high infection prevalence of intestinal parasites detected in children from R.A.A.N. is worth mentioning. In fact, it is the highest detected in Nicaragua, with the municipality of Puerto Cabezas standing out. The results obtained are likely to be the consequence of drinking and eating contaminated water and food, habits of playing or handling infested soil, eating with soiled hands, and unhygienic toilet practices. All of this is another clear example of the typical scenario of intestinal parasites present in LAC countries considered climatically suitable for parasite transmission. Besides, the presence of commensal protozoan species is important since they are indicators of poor hygienic-sanitary conditions and that have transmission mechanisms identical to the pathogenic species. Blastocystis spp. and T. trichiura were detected as the predominant enteropathogens among children from R.A.A.N., just as in previous Nicaraguan studies [18][19][20][21] and other countries [24][25][26] . However, in other regions of Nicaragua, G. intestinalis and A. lumbricoides were the most common parasites found 1,27 .
Focusing on the two studied municipalities, significant differences appear with helminth infection mainly in Puerto Cabezas, while in Siuna protozoan infection is higher. In addition to the scarce knowledge and awareness of parasitic infections 24,28,29 and the similar hygienic-sanitary conditions in both municipalities, it could be the climatic conditions that mark the differences. Although these two municipalities are separated by only about 210 km, the climate is different between them: Puerto Cabezas presents a tropical rainforest climate, while Siuna is characterized by a tropical monsoon climate. Besides, Puerto Cabezas is a coastal, more urbanized, municipality than Siuna, which is located in the interior and basically rural. This fact indicates that results from one geographical setting might not be applied to other nearby, and thus, climatic conditions, surface temperature and humidity could be more favorable for the survival of one parasite or another.
Each of the infective stages of STH (eggs of T. trichuris and A. lumbricoides; infective larvae of hookworm) has its climatic thresholds for growth and development. In fact, altered climatic parameters due to climate change are bound to influence the biological development of STH.
The high relative humidity of Puerto Cabezas throughout the entire year could be one of the causes of the higher viability of STH eggs 30 showing the dominance of A. lumbricoides and T. trichiura. Both species are more susceptible to increasing temperature, while in the case of Hookworm the trend is contrary and the higher prevalence rates are found in Siuna, more related to the fact that the prevalence of infection is apparently more susceptible to increasing desiccation besides a decreasing level of urbanization 26,31 .
Due to the climatic variability, we found that human helminthiases present a heterogenic distribution in children depending on where exactly they live, even within the same region of Nicaragua (R.A.A.N.). Likewise, significant regional differences in the prevalence and intensity of STH infections have been found in other LAC countries such as Ecuador 38 and Paraguay 32 .
Similar to other LAC countries 25,32 , no differences of infection between boys and girls were found. However, prevalence of infection was directly related to age, being highest in the 6-11-year-old group, related to starting playing outdoors and getting exposed to infections 26 . Moreover, polyparasitism, the most common form of infection, was also age-related. These results agree with previous work in Nicaragua 27,28 and other countries 11,34 .
Disease caused by STH is directly related to infection intensity. Although prevalence is used in STH epidemiological surveys, intensity of infection is more important as a determinant of morbidity 35 . The light intensity of T. trichiura is normal in endemic areas where most students harbor low or moderate burdens, while only a few individuals harbor a heavy burden 19,33,36 . Moreover, the moderate intensity of A. lumbricoides appears mostly in children of 6-11 years of age. In agreement with our results, surveys from other parts of the world also found light intensities of T. trichiura and hookworm infections, while values for A. lumbricoides were of light and moderate intensities 35 .
Adequate sanitation facilities may be important when explaining the spatial variation of helminth infection prevalence 11 . The odds of STH infection among children of R.A.A.N. who had safe water sources at their disposal were lower compared to those who had to rely on unsafe water sources (rain water). This finding corroborates previous LAC study 37 and it is in line with the result of a systematic review and meta-analysis which stated that "access to piped water was associated with lower odds of A. lumbricoides and T. trichiura infection" 38 , as having a water tap means easy access to water for hygienic practices.
The odds of STH infection increase with age since there is an age-related change in exposure to STHs, considering that as they get older they are able to play on their own in outdoor activities. This trend was also observed in other studies conducted elsewhere 39 .
There has been an increase in attention to wearing shoes for preventing soil-related disease conditions as well as STH infection. Previous research estimates that shoe-wearing lowers the odds of Hookworm infection 40,41 . The prevalence of Hookworm infection in our work is not the most commonly STH infection detected, but the lack of shoes is actually being a surrogate marker of lower socioeconomic status, which therefore increases the odds of total STH infection.
Effective strategies for controlling STH infection would have to center on reducing risk factors together with regular deworming, as anthelminthic drugs kill the parasite within the host but do not prevent reinfections and will not be sufficient to bring prevalence to low levels 35,42 . In accordance with WHO guidelines 8 , the children from R.A.A.N. are exposed to a high risk of STH infection (prevalence of any STH > 50%) and should thus continue to be treated with anthelmintics twice a year. Moreover, there is a need to increase people's knowledge on proper and environmental hygiene, mainly in those communities with lack of water sanitation and hygiene (WASH) such as Puerto Cabezas and Siuna. Results obtained in our work can be used by policy makers in Nicaragua to design geographically more specific intervention programs to reduce STH infections. www.nature.com/scientificreports/ Collaborations between research disciplines and between researchers and workers in the field responsible for individual and community health (e.g. parasitology, biochemistry, molecular biology, epidemiology, public health, etc.) is imperative to improve outcomes for people at risk from these STH infections.
The results of our study should be interpreted in the light of some limitations, mainly: only two municipilaties have been studied out of the eight that comprise R.A.A.N.; the used of Kato-Katz technique, which can miss some egg count in an area with light-intensity infections; the prevalence obtained for S. stercoralis larvae may not be considered definitive because the specific etiological technique for the detection of this nematode was not applied; and also, the low number of completed questionnaires, as well as the veracity of their responses.

Data availability
All the material analyzed as well as the database is available in the Laboratory of Parasitology of the University of Valencia from the corresponding author on reasonable request.